The present invention relates to a method of blocking the SEC receptor and also inhibiting neurotoxicity caused by amyloid-.beta. protein, a peptide that has been implicated in Alzheimer's disease and is known to bind to the SEC receptor. More particularly, the invention relates to inhibiting the neurotoxic effects of amyloid-.beta. protein with a synthetic peptide that blocks the SEC receptor. Note: Literature references on the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art and other such state-of-the-art techniques as used herein are indicated in parentheses and appended at the end of the specification, i.e. (1) to (21).
The serpin-enzyme complex (SEC) receptor recognizes a highly conserved domain in the carboxyl-terminal fragment of .alpha.,-anti-trypsin (.alpha.1-AT) and several other serpins (1). The SEC receptor-binding domain of the serpins is only available for receptor recognition after the serpin has undergone a structural rearrangement. For instance, the SEC receptor recognizes serpins after they have formed a covalently-stabilized, inhibitory complex with a cognate serine-type enzyme. The SEC receptor also recognizes serpins which have been proteolytically modified by action of metalloenzymes or, in the case of .alpha.1-AT, by the collaborative action of reactive oxygen intermediates and serine-type enzymes. The SEC receptor was originally discovered because it mediated an increase in synthesis of .alpha.1-AT in response to .alpha.1-AT-elastase complexes. Thus, this receptor was shown to be involved in a feed-forward servomechanism which allows for inhibition of the enzymatic activity and, in turn, signalling to the cell to make more inhibitor. Presumably this prevents extensive destruction of surrounding tissue during the migration of phagocytes or the sprouting of cell processes as well as during wound repair. The SEC receptor has since been shown to mediate chemotaxis of neutrophils in response to serpin-enzyme complexes (2). The SEC receptor also internalizes its ligand by classical receptor-mediated endocytosis and delivers the ligand to an acidic compartment, probably lysosome, for intracellular degradation (3). Because its ligand specificity is similar to that for in vivo clearance/catabolism of serpin-enzyme complexes, it is also probably involved in in vivo clearance/catabolism of these complexes. Photoaffinity cross-linking studies and ligand-affinity chromatographic purification studies have shown that the receptor has an .about.80 kDa ligand binding subunit (4). It is expressed in many cell types including hepatocytes, mononuclear phagocytes, neutrophils, epithelial cells, fibroblasts and cells of neuronal origin.
One interesting aspect of this receptor became apparent when the SEC receptor-binding domain of .alpha.1-AT was mapped in fine detail. A pentapeptide neodomain in the carboxyl terminal fragment of .alpha.1-AT was found to be sufficient for binding to the SEC receptor (5). See also U.S. Pat. No. 5,175,253. The interaction between the pentapeptide and the receptor was specific for length and sequence as shown by studies with deletions, substitutions and scrambling. The pentapeptide is a highly conserved region among the serpin family, thus explaining cross-competition for binding to the SEC receptor by several serpin-enzyme complexes, including .alpha.1-ACT-cat G, ATIII-thrombin, HCII-thrombin and, to a lesser extent, C1 inhibitor-C1s and PAI 1-TPA. This pentapeptide was also identified in the tachykinins and there was a similar sequence within the amyloid-.beta. which had been implicated in toxic effects on neurons(6).
Amyloid-.beta.is a major proteinaceous constituent of the extracellular deposits found in Alzheimer's disease (AD), and Down Syndrome (7). Most of the evidence suggests that amyloid-.beta. is generated by abnormal proteolytic processing of the amyloid precursor protein (APP), which leads to extracellular deposition and represents the primary pathophysiologic element of these conditions. Several recent studies have implicated a neurotoxic effect for amyloid-.beta. (6) or cores of amyloid plaques (8) by administration to cultured neurons or by injection into the brains of experimental animals. In one series of studies these effects were attributed to amyloid-.beta. 25-35 and were blocked by substance P (6). In other studies, amyloid-.beta. 25-35 was shown to enhance the neurotoxicity of glutamate (9-10). This effect of amyloid-.beta. was elicited at a half-maximal concentration of .about.40 nM and was not elicited by a peptide in which the sequence of the peptide had been reversed.
Because interaction of peptide ligands with the SEC receptor is half-maximally saturated at 40-50 nM and is sequence-specific, and because the SEC receptor is expressed on cells of neuronal origin (PC12), it constitutes an excellent candidate for mediating the neurotoxic effect of amyloid-.beta.. Therefore, it was of some interest to the present inventor and colleagues to examine the possibility that the amyloid-.beta. could bind to the SEC receptor. It has now been shown that amyloid-.beta., substance P and several other tachykinins bind to the SEC receptor (4). Amyloid-.beta., serpin-enzyme complexes, and peptide ligands for the SEC receptor do not bind to the substance P receptor. The SEC receptor is clearly distinct from the substance P receptor by several criteria:
First, the SEC receptor binds its ligands at a significantly lower affinity (K.sub.d =50 nM vs 1 nM) and is expressed in greater number of plasma membrane molecules per receptor-bearing cell (.about.450,000 vs. 10,000-20,000).
Second, the SEC receptor is much less restricted in the specificity with which it recognizes ligand as compared to the substance P receptor. The SEC receptor recognizes peptide 105Y, substance P, substance K, and neurokinin B with approximately similar affinities, whereas the substance P receptor recognizes substance P with an affinity that is 2-3 orders of magnitude higher than that for substance K or neurokinin B. Moreover, the substance P receptor does not recognize peptide 105Y, .alpha.1-AT-protease complexes, bombesin, or amyloid-.beta.. These two lines of evidence also make it likely that the SEC receptor is distinct from the substance K receptor and the neurokinin B receptor. The SEC receptor also recognizes its peptide ligands equally well with or without a C-terminal carboxyl-amide whereas the tachykinin receptors are markedly restricted in favor of a C-terminal carboxyl-amide.
A third line of evidence demonstrating the distinction between the SEC receptor and the substance P receptor is the absence of substance P receptor gene expression in HepG2 cells and human liver, known sites of expression of the SEC receptor. Thus, the SEC receptor, rather than the substance P receptor or other tachykinin receptors, is a prime candidate for mediating the neurotrophic/neurotoxic effects of amyloid-.beta..
Because data had shown that the SEC receptor mediated the neutrophil chemotactic effect of .alpha.1-AT-elastase complexes, it was predicted that amyloid-.beta. and substance P would have neutrophil chemotactic effects. This prediction was shown to be correct (2). Even more importantly, studies of the neutrophil chemotactic effects of these peptides showed that substance P elicited homologous desensitization of the SEC receptor on neutrophils to the chemotactic effects of amyloid-.beta.. Desensitization of the SEC receptor by substance P is, therefore, a possible explanation for the previous observation that substance P inhibits the trophic/toxic effects of amyloid-.beta.on neurons (6).
In recent studies, the present inventor and colleagues have examined the possibility that the SEC receptor is expressed on cells of neuronal origin. The results confirm the original observation that the NGF-responsive, ganglionic cell line PC12 bears abundant SEC receptor. In addition, expression of the SEC receptor has been demonstrated in the human glioblastoma lines U138MG and U373MG and in the human astrocytoma cell line STTG-1.
In addition to the findings which suggest that the SEC receptor mediates trophic/toxic effects on neurons, there are several other possible ways in which the SEC receptor may be involved in the secondary pathophysiological events that lead to dementia in AD:
First, it is possible that the SEC receptor plays a role in the local inflammatory response associated with amyloid-.beta. deposition. Although it has not been emphasized in the description of AD, several studies have reported an inflammatory response surrounding amyloid plaques. In studies of immune-associated antigens in human post-mortem samples, several reports show that expression of HLA-DR major histocompatibility antigen on microglial cells adjacent to amyloid plaques (11). Moreover, microglial cell proliferation and scavenging activity as well as T cell infiltration was reported at plaque sites (12). Because the SEC receptor has been demonstrated on the surfaces of cells of myeloid lineage, including monocytes and neutrophils, and because the SEC receptor has been demonstrated on the surface of human glioblastoma cell lines, as described above, it is possible that it is expressed on microgila in vivo. Moreover, it becomes a formal possibility that interaction of a ligand such as amyloid-.beta. with the SEC receptor plays a role in the directed migration of microgila, as it does for neutrophils, or in the expression of pro-inflammatory and/or anti-inflammatory products by microglia. Several studies have indicated that molecules which are known ligands for the SEC receptor elicit production of pro-inflammatory and anti-inflammatory cytokines. Kurdowska and Travis showed that .alpha.1-antichymotrypsin-protease complexes and proteolytically modified .alpha.1-antichymotrypsin mediated an increase in production of IL-6 by fibroblasts (13). Although it has not yet been studied formally, this effect is likely to be mediated by the SEC receptor. .alpha.1-antichymotrypsin-catG complexes and proteolytically modified .alpha.1-ACT cross-compete for binding of .alpha.1-AT-elastase complexes to HepG2 cells (14), and desensitize neutrophils to the chemotactic effect of .alpha.1-AT-elastase complexes (15).
Tilg et al have recently shown that peptide 105C (pentapeptide which binds to the SEC receptor), but not peptide 105C--C (tetrapeptide with deletion of C-terminal amino acid of peptide 105C, which does not bind to the SEC receptor) mediates induction of interleukin-1 receptor antagonist (IL-1RA) and interleukin-1 (IL-1.beta.) in peripheral blood mononuclear cells, with 5-to 10-fold greater induction of IL-1RA than IL-1.beta. (16). IL-1RA is known to block the biological activity of IL-1.beta. (17). This may be particularly important in AD in that IL-1.beta. has been shown to mediate increases in synthesis of the amyloid precursor protein (18), and therein, could increase the tendency toward amyloid-.beta. deposition in a susceptible genetic background.
Second, the SEC receptor may mediate ingrowth of neurites into the amyloid plaques. The SEC receptor is expressed on PC12 neuronal cells and is capable of mediating directed migration of cells such as neutrophils. An effect of this type on neurons is very important since ingrowth and degeneration of neurites is considered one of the hallmarks distinguishing mature plaques, associated with dementia, and diffuse plaques, associates with aging.
Taken together, these considerations suggest that the SEC receptor plays a role in this pathophysiology of pre-senile dementia associated with AD and that pharmacologic blockage of this receptor can be used to prevent formation of mature plaques, and, in turn, neuronal degeneration and dementia.
Although scientific explanation of the role of the SEC receptor is described herein, it is to be understood that the inventor is not limited to any particular scientific theory.